Human hematopoietic chimerism and NK cell development in HIS mice
Human HSCs engrafted into newborn BALB/c Rag2−/−
mice develop into mature myeloid and lymphoid cells (15
). We used this approach to investigate human NK cell development in vivo.
8–12 wk after HSC engraftment, HIS mice displayed human hematopoietic chimerism in all organs analyzed, with human thymopoiesis, B lymphopoiesis, and myelopoiesis evident (, and Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20082013/DC1
). Using an NK cell–specific antibody (anti-NKp46), we identified human NK cells in all lymphoid organs of HIS mice, although at low frequencies, typically between 0.3 and 1.5% of human lymphocytes (). Both CD56hi
NK cells subsets are present, with the majority of NK cells having the latter phenotype (). Interestingly, some CD16+
cells expressed CD56 at levels indistinguishable from non–NK cells. These CD56lo
cells are NK cells, as they express NKp46, NKG2D, CD94, and, like their counterparts in man, KIRs, whereas CD56hi
cells are rarely KIRs+
cells present a phenotype consistent with peripheral NK cells in man including expression of CD122 (IL-2Rβ), NKG2A, CD161, but not CD3 (). In vivo–generated human NK cells express high levels of intracellular granzyme B and IFN-γ when stimulated with IL-12 and -18 ex vivo, and they degranulate when co-cultured with K562 human leukemia cells as determined by expression of CD107a ().
Figure 1. NK cells develop and populate various lymphoid tissues in HIS mice. (A) 8 wk after CD34+CD38− HSC engraftment, various organs from HIS mice were analyzed for human NK cell reconstitution by flow cytometry (human CD45; hCD45). FACS plots (more ...)
IL-15 is a pleiotropic cytokine essential for mouse NK cell development (10
). Given the sparse number of human NK cells, we hypothesized that IL-15 availability in this HIS model might be suboptimal. HIS mice represent a hybrid human–mouse system where cytokine receptor compatibility between species may not exist. Specifically, although human IL-15 (hIL-15) induces survival and proliferation of mouse NK cells (17
), it is not clear whether the reverse is true. This is a critical question, as we expect most of the IL-15 in HIS mice to be mouse-derived given the low human myeloid (15
) or absent epithelial/stromal cell chimerism (cells likely to be sources of IL-15) (10
). We found that human NK cells cultured in vitro with hIL-15 proliferated extensively (predominately the CD56hi
subset); this was in contrast to those cultured in mouse IL-15 (mIL-15), which itself sufficiently induced proliferation of mouse NK cells ( and Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20082013/DC1
). Given that mIL-15 is trans-presented in vivo, we investigated the effect of culturing human NK cells with low concentrations of IL-15 alone or IL-15 trans-presented by IL-15Rα-Fc of the same or alternate species. Irrespective of which species of IL-15Rα was used, mIL-15 failed to induce human NK cell proliferation, although some cells survived when mIL-15 was combined with hIL-15Rα-Fc (). In contrast, hIL-15Rα was clearly superior in inducing NK cell proliferation compared with mIL-15Rα when combined with hIL-15 (). Similarly, activated human, but not mouse, myeloid cells were able to induce human NK cell proliferation in vitro (Fig. S2). Last, human NK cells are observed in athymic HIS mice (Rag2−/−
), ruling out a major role for T cell–derived IL-2 driving NK cell development ().
Collectively, these data indicate that the few resident NK cells in HIS mice are dependent on the available hIL-15. Indeed, the number of human NK cells in the spleen of HIS mice could be further reduced after treatment with neutralizing antibody against human, but not mouse, IL-15 (Fig. S2). Furthermore, hIL-15Rα+
cells known to express IL-15 and support NK cell development, such as members of the myeloid lineage, are found in the bone marrow of HIS mice (unpublished data) (10
). Given this last point, it is possible that enhancing the chimerism of hIL-15/-15Rα–expressing cells will improve human NK cell development in HIS mice.
Given the likely dependence of human NK cells on hIL-15, we next treated HIS mice with the same concentration of hIL-15 alone or hIL-15 preincubated with hIL-15Rα-Fc to mimic IL-15 trans-presentation in vivo. Consistent with mouse studies (11
), we observed a significant increase in NK cell frequency when hIL-15 was complexed to IL-15Rα-Fc, but not when administered alone (). Increased NK lymphopoiesis by hIL-15+IL-15Rα-Fc compared with IL-15 alone was associated with increased Bcl-xL expression and cell proliferation, as demonstrated by BrdU uptake and Ki-67 expression (
). These data indicate that IL-15R–mediated human NK cells responses are more readily evoked in vivo when IL-15 is complexed to IL-15Rα-Fc.
Figure 2. Trans-presentation of hIL-15 promotes human NK cell homeostasis in vivo. (A) HIS mice were injected i.p. (every 5 d for 15 d) with hIL-15, IL-15+IL-15Rα-Fc, or PBS commencing 6 wk after reconstitution. 3 d after the last injection, bone (more ...)
We next compared two different IL-15R agonists, both mimicking IL-15 trans-presentation, hIL-15+IL-15Rα-Fc, and RLI (hIL-15 covalently linked to an hIL-15Rα extended sushi domain, but lacking any Fc fragment) (11
). Four injections of 2.5 μg hIL-15R agonists (one per week) resulted in a significant increase in the number of NKp46+
cells in all organs (). Both these agonists markedly enhanced NK cell in vivo uptake of BrdU; expression of Bcl-xL, Ki-67, and CD69; and resulted in delayed apoptosis of NK cells when withdrawn from cytokines (, and not depicted), suggesting both proliferation and survival result from IL-15+IL-15Rα binding to NK cells in vivo. Human CD8 T cells were also significantly augmented in HIS mice and had an obvious increase in BrdU uptake after treatment with RLI (). Interestingly, RLI that activates both the IL-15Rβ/γ and IL-15Rα/β/γ was consistently more effective in vivo than noncovalent association of hIL-15+hIL-15Rα-Fc that activates IL-15Rβ/γ alone. This observation is in agreement with in vitro studies showing RLI functions more efficiently than the noncovalent associations of IL-15 + sushi domain or other soluble forms of IL-15R (20
). The evident effect of RLI compared with IL-15+IL-15Rα-Fc also indicates that the Fc protein does not contribute to the augmented NK lymphopoiesis and rules out any activation of human NK cells through CD16.
Although IL-15 is known to heighten NK cell cytotoxicity and induce proliferation, it also protects cells from apoptosis (the latter possible at lower concentrations) by suppressing proapoptotic Bim and elevating Bcl-2 family members such as Bcl-xL and Mcl-1 (18
). A clear in vivo effect of RLI and IL-15+IL-15Rα-Fc was the up-regulation of Bcl-xL in NK cells (). We next asked if enhanced survival could improve NK cell reconstitution in the limiting hIL-15 environment of HIS mice. To address this, HSCs were infected with a bicistronic retrovirus encoding the pro-survival protein Bcl-xL and GFP (to detect infected cells) in vitro before engrafting newborn BALB/c Rag2−/−
mice. Ectopic expression of Bcl-xL in human HSCs resulted in a significant increase in thymocyte and splenocyte cellularity 8 wk after engraftment, with Bcl-xL–transduced cells (GFP+
) representing a greater proportion of hCD45+
cells compared with control transduced cells in all organs (). NKp46+
cells were significantly increased in thymus and spleen of Bcl-xL HIS mice compared with control HIS mice; however, this appeared to be primarily a result of increased cellularity in these organs as the percentage of NK cells largely unchanged (). In addition, although a greater proportion of NK cells were GFP+
in Bcl-xL–infected mice compared with controls, no accumulation of Bcl-xL–expressing NK cells was observed among the most mature subset (CD56lo
). Lastly, the number of NK cells in HIS mice engrafted with Bcl-xL–infected HSCs, in terms of absolute numbers and fold difference compared with control, were substantially lower than mice treated with trans-presented IL-15. These findings indicate that IL-15/IL-15Rα effects extend beyond providing survival signals in promoting human NK cell development in vivo.
Figure 3. Ectopic expression of Bcl-xL mildly augments human NK cell reconstitution in vivo. Human fetal liver HSCs were infected with retrovirus encoding hBcl-xL and GFP or GFP alone and used to generate HIS mice. (A) 8 wk after engraftment, thymus, spleen, and (more ...)
A consistent observation in all lymphoid organs after treatment with trans-presented IL-15 was the skewing of NK cell maturation toward the more differentiated CD56lo
phenotype. In particular, we observed significant and specific increases in the frequency of CD16+
NK cells and the ratio of CD16+
NK cells 7 d after the final treatment (). This in vivo accumulation is consistent with a model where CD56lo
NK cells represents the terminal stage of NK cell development and suggests that trans-presented IL-15 promotes this differentiation. CD16 expression is associated with human NK cell differentiation and increased cytotoxicity by means of increased intracellular effector granules and the ability to perform antibody-dependent cell cytotoxicity. IL-15 itself is known to enhance NK cell cytotoxicity via up-regulating effector molecules, such as IFN-γ, perforin, and granzymes (24
). Strikingly, among the enhanced CD16+
population after IL-15 trans-presentation treatment, the percentage of NK cells expressing KIRs was also significantly elevated, resulting in a large increase in the KIR+
NK cell pool, an effect that is also more prominent with RLI (). The fraction of KIR+
NK cells after exogenous IL-15 trans-presentation was typically highest in the thymus and elevated compared with normal frequencies in human blood (typically 50–60%). This may result from greater IL-15 concentrations in our model compared with humans; however, the frequency of KIR+
NK cells in human thymus has not been reported using all existing commercial antibodies to KIRs. In contrast to IL-15 trans-presentation, no enhanced NK cell differentiation and induction of KIR expression was observed in HIS mice treated with IL-15 alone or in those engrafted with Bcl-xL–expressing HSCs (). Furthermore, the increase in KIR+
NK cells did not represent an expansion of one CD56lo
NK clone, as CD56lo
NK cells expressing a combination of 1–5 different KIR members were present in this population ().
Figure 4. HIL-15R agonists promote NK cells differentiation in vivo. (A) NK cell maturation and KIR expression was analyzed in thymus, bone marrow, and spleen of HIS mice treated as in by flow cytometry for surface expression of CD56, CD16, and KIRs (KIR-2DL2/3/1/-2DS1/2/4/-3DL1/S1). (more ...)
It is unlikely the accumulation of CD56lo
NK cells results from a preferential expansion of this subset in response to hIL-15 trans-presentation as both CD56lo
NK cell subsets had a similar level of incorporated BrdU during treatment (). Although one cannot distinguish between CD16+
NK cells that have incorporated BrdU and BrdU+
cells that have up-regulated CD16, it is most likely that the latter accounts for most of the BrdU+
NK cells as CD56lo
NK cells are refractory to IL-15 stimulation in vitro (25
), especially when compared with CD56hi
NK cells (). To test this hypothesis, we sorted and transferred either highly purified CD56hi
NK cells from fetal spleen into BALB/c Rag2−/−
in the presence or absence of RLI treatment. Although we were unable to recover the donor cells in mice receiving PBS alone, clear populations of donor cells were recovered from RLI-treated mice 7 d after transfer (). Consistent with our hypothesis, trans-presented IL-15 induced the differentiation of CD56hi
NK cells, with between 34 and 40% of recovered NK cells becoming CD16+
(). In addition a fraction of the CD16+
NK cells also now expressed KIRs. Similarly, whereas CD56lo
NK cells retained this phenotype in the presence of RLI after transfer, expression of KIRs were acquired by a fraction of these CD56lo
NK cells that lacked KIR expression before transfer (). The absence of NK cells in non-RLI treated recipients clearly shows that the available mouse IL-15 is inadequate to support human NK cell survival and highlights the importance of a source of hIL-15 in HIS mice in promoting NK cell differentiation.
Figure 5. IL-15-dependent development of CD16+KIR+ NK cells from CD16−KIR− precursors in vivo. (A) NK cell subsets in the bone marrow of RLI-treated HIS mice from were analyzed for BrdU uptake using flow cytometry. FACS (more ...)
Binding of IL-15 to NK cells ultimately activates signaling pathways stemming from STAT5 phosphorylation, such as activation of NF-κB, induction of cyclin D, down-regulation of proapoptotic proteins, and up-regulation of Bcl-2 family members and cytolytic granules (18
). Although it is clear how these signaling events promote cell division, survival, and effector functions, it is not known how these pathways regulate acquisition of KIRs. Coordination between granzyme/perforin induction and KIR expression would be one means to limit NK cell activity during differentiation, and whereas most NK cells express other inhibitory receptors for MHC-I (CD94/NKG2A and LILR) throughout development, it appears acquisition of KIRs is a late event and likely unique compared with other forms of MHC-I–mediated inhibition. Collectively, these data provide the first in vivo demonstration that trans-presented IL-15 is essential for NK cell survival and subsequent differentiation and that KIR expression on mature NK cells is dynamically regulated by IL-15 signaling.